p53 Deficiency rescues neuronal apoptosis but not differentiation in DNA polymerase beta-deficient mice

In mammalian cells, DNA polymerase beta (Polbeta) functions in base excision repair. We have previously shown that Polbeta-deficient mice exhibit extensive neuronal cell death (apoptosis) in the developing nervous system and that the mice die immediately after birth. Here, we studied potential roles in the phenotype for p53, which has been implicated in DNA damage sensing, cell cycle arrest, and apoptosis. We generated Polbeta(-/-) p53(-/-) double-mutant mice and found that p53 deficiency dramatically rescued neuronal apoptosis associated with Polbeta deficiency, indicating that p53 mediates the apoptotic process in the nervous system. Importantly, proliferation and early differentiation of neuronal progenitors in Polbeta(-/-) p53(-/-) mice appeared normal, but their brains obviously displayed cytoarchitectural abnormalities; moreover, the mice, like Polbeta(-/-) p53(+/+) mice, failed to survive after birth. Thus, we strongly suggest a crucial role for Polbeta in the differentiation of specific neuronal cell types.     

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)-dependent protein kinase catalytic subunit (DNA-PKcs)-null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice.     

Elevated DNA double strand breaks and apoptosis in the CNS of scid mutant mice

Genetic approaches have provided evidence that DNA end-joining problems serve an essential role in neuronal survival during development of mammalian embryos. In the present study, we tested whether the DNA repair enzyme, DNA dependent protein kinase, plays an important role in the survival of cerebral cortical neurons in mice. DNA-PK is comprised of a DNA-binding subunit called Ku and a catalytic subunit called DNA-PKcs. In mice with the scid mutation, DNA-PKcs is truncated near the kinase domain, which causes loss of kinase activity. We compared the spatial and temporal aspects of neuronal cell death in scid versus isogenic wild-type embryos and found a significant increase in dying cells in scid mice, as assessed by nuclear changes, DNA fragmentation and caspase-3 activity. Additional biochemical and immunocytochemical studies indicated that of several DNA repair enzymes investigated, only PARP was increased in scid mice, possibly in response to elevated DNA strand breaks.     

p53-Independent apoptosis disrupts early organogenesis in embryos lacking both ataxia-telangiectasia mutated and Prkdc

The ataxia-telangiectasia mutated (ATM) protein and the nonhomologous end-joining (NHEJ) pathway play crucial roles in sensing and repairing DNA double-strand breaks in postnatal cells. However, each pathway is dispensable for early embryogenesis. Loss of both ATM and Prkdc/Ku is synthetically lethal, but neither the developmental processes perturbed nor the mechanisms of lethality have been determined by previous reports. Here, we show that ATM and Prkdc collaborate to maintain genomic stability during gastrulation and early organogenesis, a period of rapid proliferation and hypersensitivity to DNA damage. At E7.5 to E8.5, ATM(-/-)Prkdcscid/scid embryos displayed normal proliferation indices but exhibited excessive apoptosis and elevated expression of Ser15-phosphorylated p53. Thus, this crucial regulatory residue of p53 can be phosphorylated in the absence of ATM or Prkdc. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that apoptosis is p53 independent in these in ATM(-/-)Prkdcscid/scid embryos. Because mice with combined disruptions of ATM and other NHEJ components (ligase IV, Artemis) are viable, our data suggest a novel NHEJ-independent function for Prkdc/Ku that is required to complete early embryogenesis in the absence of ATM.     

DNA-dependent protein kinase catalytic subunit is not required for dysfunctional telomere fusion and checkpoint response in the telomerase-deficient mouse

Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerase-deficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc(-/-) DNA-PKcs(-/-) cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)--an additional crucial NHEJ component--was also permissive for chromosome fusions in mTerc(-/-) cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNA-PKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc(-/-) tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.     

Role of DNA-dependent protein kinase in neuronal survival

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.     

Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase beta

DNA polymerase beta (Polbeta) has been implicated in base excision repair in mammalian cells. However, the physiological significance of this enzyme in the body remains unclear. Here, we demonstrate that mice carrying a targeted disruption of the Polbeta gene showed growth retardation and died of a respiratory failure immediately after the birth. Histological examination of the embryos revealed defective neurogenesis characterized by apoptotic cell death in the developing central and peripheral nervous systems. Extensive cell death occurred in newly generated post-mitotic neuronal cells and was closely associated with the period between onset and cessation of neurogenesis. These findings indicate that Polbeta plays an essential role in neural development.     

KU70/80, DNA-PKcs, and Artemis are essential for the rapid induction of apoptosis after massive DSB formation

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.     

Defective chromatin recruitment and retention of NHEJ core components in human tumor cells expressing a Cyclin E fragment

Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.     

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.     

Function of DNA-protein kinase catalytic subunit during the early meiotic prophase without Ku70 and Ku86

All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction. Despite the fact that early spermatocytes lack the Ku proteins, spontaneous apoptosis of these cells occurred in the scid testis. The majority of these apoptotic spermatocytes were found at stage IV of the cycle of the seminiferous epithelium where a meiotic checkpoint has been suggested to exist. Meiotic synapsis and recombination during the early meiotic prophase induce DSBs, which are apparently less accurately repaired in scid spermatocytes that then fail to pass the meiotic checkpoint. The role for DNA-PKcs during the meiotic prophase differs from that in mitotic cells; it is not influenced by ionizing radiation and is independent of the Ku heterodimer.     

DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage.Five major DNA repair pathways are homologous recombinational repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). In each of these DNA repair pathways, key proteins occur with dual functions in DNA damage sensing/repair and apoptosis. Proteins with these dual roles occur in: (1) HRR (BRCA1, ATM, ATR, WRN, BLM, Tip60 and p53); (2) NHEJ (the catalytic subunit of DNA-PK); (3) NER (XPB, XPD, p53 and p33(ING1b)); (4) BER (Ref-1/Ape, poly(ADP-ribose) polymerase-1 (PARP-1) and p53); (5) MMR (MSH2, MSH6, MLH1 and PMS2). For a number of these dual-role proteins, germ line mutations causing them to be defective also predispose individuals to cancer. Such proteins include BRCA1, ATM, WRN, BLM, p53, XPB, XPD, MSH2, MSH6, MLH1 and PMS2.     

A role for endogenous and radiation-induced DNA double-strand breaks in p53-dependent apoptosis during cortical neurogenesis

Prenatal exposure to low-dose radiation increases the risk of microcephaly and/or mental retardation. Microcephaly is also associated with genetic mutations that affect the non-homologous end-joining pathway of DNA double-strand break repair. To examine the link between these two causal factors, we characterized the neural developmental effects of acute radiation exposure in mouse littermate embryos harboring mutations in the Ku70 and p53 genes. Both low-dose radiation exposure and Ku70 deficiency induced morphologically indistinguishable cortical neuronal apoptosis. Irradiated Ku70-deficient embryos displayed anatomical damage indicative of increased radiosensitivity in the developing cerebral cortex. Deleting the p53 gene not only rescued cortical neuronal apoptosis at all levels but also restored the in vitro growth of Ku70-deficient embryonic fibroblasts despite the presence of unrepaired DNA/chromosomal breaks. The results confirm the role of DNA double-strand breaks as a common causative agent of apoptosis in the developing cerebral cortex. Furthermore, the findings suggest a disease mechanism by which the presence of endogenous DNA double-strand breaks in the newly generated cortical neurons becomes radiomimetic when DNA end joining is defective. This in turn activates p53-dependent neuronal apoptosis and leads to microcephaly and mental retardation.     

Mutations in the p53 and scid genes do not cooperate in lymphomagenesis in doubly heterozygous mice

Analysis of double mutant mice of the p53 and scid genes, which have a combination of cell cycle checkpoint/apoptosis and DNA repair defects, shows that the latter defect synergistically enhances lymphoma development with loss of the former function. These mice lack the ability to eliminate lymphocytes predisposed to neoplastic transformation resulting from faulty antigen receptor gene rearrangement. Here we examine the cooperativity in double heterozygotes of p53 and scid in which normal development of lymphocytes is not impaired. MSM mice carrying a p53-knockout allele were crossed with BALB/c mice heterozygous for the scid locus and 129 offspring were obtained. They were subjected to gamma-ray irradiation, 84 thymic lymphomas being generated. The tumors and host mice were genotyped of p53 and scid. Among 42 mice developing p53-deficient lymphomas, scid/+ and +/+ genotypes did not provide difference in onset and latency. Besides, allelic loss of the Scid gene occurred at a high frequency in those lymphomas but the loss exhibited no allelic bias. The results suggest that the scid/+ genotype is not a modifier of loss of p53 function in the double heterozygotes.     

Dysfunctional mammalian telomeres join with DNA double-strand breaks

In addition to joining broken DNA strands, several non-homologous end-joining (NHEJ) proteins have a second seemingly antithetical role in constructing functional telomeres, the nucleoprotein structures at the termini of linear eukaryotic chromosomes that prevent joining between natural chromosome ends. Although NHEJ deficiency impairs double-strand break (DSB) repair, it also promotes inappropriate chromosomal end fusions that are observed microscopically as dicentric chromosomes with telomeric DNA sequence at points of joining. Here, we test the proposition that unprotected telomeres can fuse not only to other dysfunctional telomeres, but also to ends created by DSBs. Severe combined immunodeficiency (scid) is caused by a mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-PK), an enzyme required for both efficient DSB repair and telomeric end-capping. Cells derived from wild-type, Trp53-/-, scid, and Trp53-/-/scid mice were exposed to gamma radiation to induce DSBs, and chromosomal aberrations were analyzed using a novel cytogenetic technique that can detect joining of a telomere to a DSB end. Telomere-DSB fusions were observed in both cell lines having the scid mutation, but not in wild-type nor Trp53-/- cells. Over a range of 25-340 cGy, half of the visible exchange-type chromosomal aberrations in Trp53-/-/scid cells involved telomere-DSB fusions. Our results demonstrate that unprotected telomeres are not only sensed as, but also acted upon, by the DNA repair machinery as if they were DSB ends. By opening a new pathway for misrepair, telomere-DSB fusion decreases the overall fidelity of DSB repair. The high frequency of these events in scid cells indicates telomere dysfunction makes a strong, and previously unsuspected, contribution to the characteristic radiation sensitivity associated with DNA-PK deficiency.     

Akt promotes post-irradiation survival of human tumor cells through initiation, progression, and termination of DNA-PKcs-dependent DNA double-strand break repair

Akt phosphorylation has previously been described to be involved in mediating DNA damage repair through the nonhomologous end-joining (NHEJ) repair pathway. Yet the mechanism how Akt stimulates DNA-protein kinase catalytic subunit (DNA-PKcs)-dependent DNA double-strand break (DNA-DSB) repair has not been described so far. In the present study, we investigated the mechanism by which Akt can interact with DNA-PKcs and promote its function during the NHEJ repair process. The results obtained indicate a prominent role of Akt, especially Akt1 in the regulation of NHEJ mechanism for DNA-DSB repair. As shown by pull-down assay of DNA-PKcs, Akt1 through its C-terminal domain interacts with DNA-PKcs. After exposure of cells to ionizing radiation (IR), Akt1 and DNA-PKcs form a functional complex in a first initiating step of DNA-DSB repair. Thereafter, Akt plays a pivotal role in the recruitment of AKT1/DNA-PKcs complex to DNA duplex ends marked by Ku dimers. Moreover, in the formed complex, Akt1 promotes DNA-PKcs kinase activity, which is the necessary step for progression of DNA-DSB repair. Akt1-dependent DNA-PKcs kinase activity stimulates autophosphorylation of DNA-PKcs at S2056 that is needed for efficient DNA-DSB repair and the release of DNA-PKcs from the damage site. Thus, targeting of Akt results in radiosensitization of DNA-PKcs and Ku80 expressing, but not of cells deficient for, either of these proteins. The data showed indicate for the first time that Akt through an immediate complex formation with DNA-PKcs can stimulate the accumulation of DNA-PKcs at DNA-DSBs and promote DNA-PKcs activity for efficient NHEJ DNA-DSB repair.     

Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated autophosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.     

Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks

53BP1 (p53 binding protein) is a BRCT domain-containing protein that is rapidly recruited to DNA double strand breaks (DSBs). To investigate the role of 53BP1 in the DNA damage response, we generated 53BP1(-/-) cells from the chicken DT40 cell line. As in mammalian cells, mutation of 53BP1 increased cellular sensitivity to ionizing radiation. Although depletion of 53BP1 resulted in checkpoint defects in mammalian cells, DT40 53BP1(-/-) cells had normal intra S phase and G2/M checkpoints. G1 specific radiosensitivity and a higher sensitivity to topoisomerase II suggested defective non-homologous end joining (NHEJ) defects in DT40 53BP1(-/-) cells. Genetic analyses confirm this suggestion as we have demonstrated an epistatic relationship between 53BP1 and the NHEJ genes, Ku70 and Artemis, but not with Rad54, a gene essential for repair of DSBs by homologous recombination. We conclude that the major role of 53BP1 in supporting survival of DT40 cells that have suffered DNA DSBs is in facilitating repair by NHEJ.     

UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.

DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion.     

Shortened telomeres in murine scid cells expressing mutant hRAD54 coincide with reduction in recombination at telomeres

Murine severe combined immunodeficiency (scid) cells are characterized by defective Prkdc (DNA-PKcs), one of the key genes involved in the repair of DNA double-strand breaks. Interestingly, scid mice are not null mutants and their cells are likely to show low DNA-PKcs activity. Prkdc is also involved in telomere maintenance and in contrast to mice genetically engineered to lack Prkdc (i.e. null mutants), which show complete absence of DNA-PKcs activity, loss of telomere capping function and normal telomere length, cells from scid mice show not only loss of telomere capping function but also abnormally elongated telomeres. Here we demonstrate that telomere elongation observed in murine scid cells can be reversed by expressing mutant hRAD54, a protein involved in homologous recombination. In addition, we measured recombination rates at telomeres using chromosome orientation fluorescence in situ hybridization (CO-FISH) and found that these are elevated in scid cells in comparison with control cells, or significantly reduced in scid cells expressing mutant hRAD54. Similarly, recombination rates at telomeres are reduced in scid cells following introduction of functional Prkdc. Since expression of mutant hRAD54 and restoration of functional Prkdc in scid cells cause the same effects, i.e. telomere shortening and reduced recombination rates at telomeres, these results argue that telomere elongation in scid cells is a complex trait resulting from interactions between homologous recombination mechanisms and DNA-PKcs.